Project Summary/Abstract from Parent Award: This work has two overall goals: (1) to understand the lifestyle of the mobile genetic elements (SCCs) that carry methicillin resistance in S. aureus, and (2) to understand the mechanism of self-loading initiator helicases, using as model systems ones that are encoded by SCC elements and their homologs from a different family of mobile elements, the SaPIs. We will address these with a combination of biochemical and structural tools, and in collaboration with Dr. Jos Penads, microbial genetics. Despite the medical relevance of SCC elements, very little is known about their molecular biology beyond the site-specific recombinases that integrate and excise them into / out of the host chromosome: no other core ?housekeeping? genes have been characterized. By examining numerous SCC elements, we defined two patterns of conserved ORFs surrounding the recombinases. Our analysis of their sequences suggests that they are novel replication modules. Both patterns include a putative helicase with a homolog among the replication initiator (?Rep?) proteins of the staphylococcal pathogenicity islands (SaPIs). The SaPIs are an otherwise-unrelated family of mobile genetic elements that are better characterized than the SCCs and are known to replicate after excision. The best-studied SaPI Rep, that of SaPIBov1, is a self-loading helicase: it recognizes and opens a bubble in an origin of replication in dsDNA, and has ATP-dependent helicase activity. We found that the SaPIBov1 Rep homolog from SCCmec type IV is an active helicase and determined its crystal structure. Surprisingly, the closest structural homolog to its ATPase domain is MCM, the archaeal / eukaryotic replicative helicase. Because these Rep proteins are easy to work with, they are excellent systems for asking how self-loading helicases morph from binding dsDNA to forming a ring around a single strand, and for understanding the mechanism of MCM-type AAA+ helicases as well. Aim 1 asks are the putative replication proteins of SCC elements functional and what exactly do they do? Preliminary results suggest that as well as the putative initiator helicases, these include novel SSBs and a minimalist PolA family polymerase that may be a primase. We will continue use biochemical tools to work out their in vitro activities and interactions. Our collaborator Dr. Penads will test their proposed functions in vivo in S. aureus. (No funds are requested for Dr. Penads). Aim 2 asks How do self-loading initiator helicases work? We will use our existing crystal structure in conjunction with electron microscopy to understand how these enzymes interact with ssDNA in helicase mode. To understand the process of bubble opening, we will combine our existing structure with DNA footprinting, DNA topology, other biochemistry and electron microscopy to model the complex that we propose is two hexamers bound to ~300bp of DNA, before and after bubble formation.